Lateral flow diagnostic devices including a micro-porous element along which a sample fluid flows laterally and a capture region for binding an analyte of interest contained in the sample fluid are known in the art. A lateral flow diagnostic device of simple construction includes a rectangular micro-porous strip, which supports capillary fluid flow along its length. Generally, quantitative and sensitive detection using such devices is limited. More recently, devices that incorporate instrumentation that allow for quantitative determination of the amount of analyte in a sample have been disclosed.
The lateral flow diagnostic strip has become widely used in assay techniques. In its simplest form the prior-art lateral flow device comprises a micro-porous strip element, which supports capillary flow of a fluid along its length. The strip has one end for application of a sample containing an analyte to be measured, a first region along its length containing a mobile reporter conjugate (typically a visually observable reporter such as colloidal gold conjugated to a first antibody directed against the analyte) and a second region containing a capture reagent (typically a second antibody directed against the analyte), and an effluent end. Sample fluid applied to one end of the strip flows along the strip to the first region where a complex is formed between the analyte and the reporter conjugate. The sample, including the mobile reporter conjugate-analyte complex, flows to the second region where the reporter conjugate-analtye complex is captured, while uncomplexed mobile reporter conjugate flows beyond the capture region towards the effluent end of the strip. The amount of visually detectable signal at the capture region is a measure of the amount of analyte in the sample. Prior art lateral flow devices are used in the above described sandwich immunoassay format as well as in an inhibition or competitive binding format.
Because prior-art lateral flow devices are inexpensive, give rapid results and are easy to use, they have been used in non-laboratory applications in so-called field-able, on-site testing or point of care diagnostic applications. Devices of the prior art have been routinely used for non-instrumented, non-quantitative diagnostic applications at the point of care, the presence of an analyte at or above a threshold concentration being determined by observing the appearance of a visible signal at the capture region. However, devices of the prior art are not generally suitable for use in quantitative assays for two reasons. Firstly, they are usually formatted with visually observable reporters, which are suitable for threshold yes/no detection, but unsuitable for quantitative analysis. Secondly, both the concentration of the complex formed between the analyte and the reporter conjugate and the amount of binding at the capture site are flow rate dependent. The variability of device operation, particularly sample flow rate and sample evaporation, creates significant variability in the detected signal.
Recently workers in the field have disclosed quantitative lateral flow devices incorporating instrumentation to measure the amount of signal at the capture site when using a chromophore reporter, or to measure the light emitted upon laser excitation of the capture region when using a fluorescent reporter (U.S. Pat. Nos. 5,753,517 and 6,497,842). U.S. Pat. Nos. 5,753,517 and 6,194,222 disclose instrumented quantitative lateral flow methods using internal controls incorporated into the flow path for internal calibration of variable factors, in particular variable flow rates. However, even quantitative prior-art lateral flow devices, have not matched the sensitivity of more complex laboratory based assays. There are three primary reasons for lower sensitivity. The first reason is the absence of rigorous wash steps, which may be required to fully remove unbound reporter conjugate from the capture region. The second reason is the absence of an amplification step. The third reason is the absence of a high sensitivity detection technique such as chemiluminescent detection. Because they are less sensitive, lateral flow devices are only used in routine analysis of higher abundance analytes. Low abundance analytes must still be measured on laboratory equipment, which incorporates rigorous wash steps, enzymatic signal amplification and extremely sensitive chemiluminescent detection techniques.
Lateral flow devices that account for some of these shortcomings are known in the prior art. U.S. Pat. No. 6,306,642 discloses a device with a primary lateral flow element for formation and capture of an enzyme-conjugate/analyte complex, and a supplementary lateral flow element containing a chromogenic substrate and a means of delaying the delivery of a chromogenic substrate to the capture region. U.S. Pat. No. 6,316,205 discloses a two-step lateral flow device with improved wash-out of unbound conjugate using a lateral flow element to which sample fluid is applied and an absorption pad separated by a removable barrier with a supplementary manual second step application of a wash fluid.
High sensitivity assays for detection of analtyes using multi-step procedures in conventional laboratory equipment are well known in the art. “Luminescence Biotechnology” eds. K. Van Dyke, C. Van Dyke and K. Woodfork, CRC Press, 2002, contains numerous examples of highly sensitive luminescence based assays. Enzyme immuno-assay kits based on membrane capture in a flow-through configuration (as opposed to lateral flow) are also known in the art. These kit-based devices typically require multiple reagent additions and wash steps and consequently are not well adapted to point-of care applications where a simple one-step procedure is preferable.
Flow-through type membrane based immunoenzymatic devices with a one-step format are now being developed. U.S. Pat. No. 5,783,401 discloses a device utilizing controlled transport membranes to provide the timed sequence of reaction steps in a multi-step enzyme immunoassay format.
Devices containing electro-osmotically pumped and pneumatically driven fluids in micro-channels (capillary dimensioned tubes, troughs and channels) are well known in the art. These devices are commonly referred to as ‘lab-on-a-chip’ devices (for example U.S. Pat. Nos. 4,908,112 and 5,180,480). Reactions, mixture separations or analyses can take place in such microstructures in liquids that are electrokinetically or pneumatically transported along conduits. However, generally in these prior art devices, reagents are stored off-chip and need to be introduced during use. Also, devices of these technologies have generally operated in a continuous flow format because valves have been difficult to construct.
Electro-osmotically pumped solid hydrophilic matrix transport paths have been disclosed in U.S. Pat. Appl. Publ. No. 2002/0179448. Self-contained devices with integral reagents featuring electro-osmotically pumped lateral flow injection into micro-reactors have been disclosed in co-pending U.S Pat. Appl. Publ. No. 20030127333. U.S Pat. Appl. Publ. No. 2002/0123059 discloses a self-contained assay device with chemiluminescence detection based on pressure driven flow in micro-channels. Lateral flow immunochromatographic devices with electrochemical detection using integral electrodes have been disclosed in U.S. Pat. No. 6,478,938.
In summary, one-step prior art lateral flow diagnostic devices lack the amplification, washing and high sensitivity detection steps needed for quantitative determination of analyte levels. Micro-channel devices in the prior art have not incorporated chemical entities in the channels and reagents storage within the device. The prior art does not teach a one-step assay device that is as easy to use and inexpensive to manufacture but which features the more advanced fluidic capability found in high sensitivity quantitative laboratory-based assay technologies and in which assay performance is largely independent of the fluidic components and reaction vessels in which the assay is performed. This invention addresses the need to adapt standard lateral flow elements to incorporate more advanced fluidic elements for use in conjugate label application, washing, amplification and enhanced sensitivity detection without sacrificing the speed, simplicity of use and low cost of standard lateral flow technologies.